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. 2012 Dec 28;304(5):G527–G535. doi: 10.1152/ajpgi.00388.2012

Fig. 3.

Fig. 3.

OA-induced cAMP formation and muscle relaxation via TGR5. A: TGR5-mediated cAMP formation. Cultured muscle cells transfected with control siRNA or TGR5-specific siRNA were treated with OA (10 μM) for 1 min, and cAMP formation was measured by ELISA. Inset: downregulation of TGR5 was determined by Western blot. B: muscle cells isolated from wild-type (WT) and tgr5−/− mice were treated with OA (10 μM) for 1 min, and the cAMP formation was measured by ELISA. Results are expressed as percent increase above basal levels. Values are means ± SE of 3 experiments. **P < 0.001 vs. control siRNA or WT. C: TGR5-mediated muscle relaxation. Dispersed gastric muscle cells from WT and tgr5−/− mice were treated with the contractile agonist carbachol (CCh, 0.1 μM) in the presence or absence of OA (10 μM) for 10 min, and the decrease in muscle cell length was measured by scanning micrometry. Decrease in muscle cell length from the basal length reflected muscle contraction, and inhibition of contractile response reflected muscle relaxation. The effect of CCh is similar in both WT (26 ± 3% decrease from basal cell length of 91 ± 3 μm) and tgr5−/− (25 ± 4% decrease in cell length of 88 ± 5 μm). OA alone had no effect on basal muscle cell length, but significantly inhibited CCh-induced sustained contraction in muscle cells from WT mice, but not in muscle cells from tgr5−/− mice. Results are expressed as % inhibition of contraction (relaxation) by OA. Values are means ± SE of 3 experiments. **P < 0.001 vs WT. D: PKA-dependent and -independent relaxation in response to OA. Dispersed gastric muscle cells were treated with the contractile agonist CCh (0.1 μM) in the presence or absence of OA (10 μM) or cAMP analog that selectively activates Epac (8-pCPT-2′-O-Me-cAMP, 10 μM) for 10 min, and the decrease in muscle cell length was measured by scanning micrometry. In some experiments the effect of OA or 8-pCPT-2′-O-Me-cAMP on CCh-induced contraction was measured in the presence of PKA inhibitor myristoylated PKI (1 μM). Relaxation was expressed as % inhibition of CCh-induced sustained contraction (28 ± 3% decrease in cell length from the basal length of 94 ± 5 μm). Values are means ± SE of 4 experiments. **P < 0.001 vs. response in the absence of myristoylated PKI.